Tryptophan hydroxylase in rat brainstem extracts is activated 2 to 2.5 fold by ATP and Mg2+ in the presence of subsaturating concentrations of the pteridine cofactor 6-methyltetrahydropterin (6MPH4). The activation by ATP-Mg2+ is also observed if the natural cofactor tetrahydrobiopterin or the synthetic cofactor dimethyltetrahydropterin is used. The activation requires ATP and Mg2+ and is not dependent on cyclic nucleotides. The ATP-Mg2+ stimulation is enhanced by calcium and can be blocked by EGTA. Removal of calmodulin from the hydroxylase containing extracts by affinity chromatography on fluphenazine-Sepharose rendered tryptophan hydroxylase unresponsive to activation by ATP-Mg2+. The readdition of calmodulin restored the ATP-Mg2+-induced activation in a calcium dependent manner. Drugs and various peptides which bind to calmodulin were found to have variable effects on the activation of tryptophan hydroxylase by phosphorylating conditions.